Biological Activities of Extracts from Ageratum fastigiatum: Phytochemical Study and In Silico Target Fishing Approach.

In the present study, the ethanolic extract from aerial parts of Ageratum fastigiatum was evaluated in vitro against epimastigote forms of Trypanosoma cruzi (Y strain), promastigote forms of Leishmania amazonensis (PH8 strain), and L. chagasi (BH400 strain). The extract was also evaluated against Staphylococcus aureus (ATCC 25 923), Escherichia coli (ATCC 11 775), Pseudomonas aeruginosa (ATCC 10 145), and Candida albicans (ATCC 36 802). The phytochemical screening was performed by thin-layer chromatography and high-performance liquid chromatography. The extract was fractionated using flash preparative chromatography. The ethanolic extract showed activity against T. cruzi, L. chagasi, and L. amazonensis and antimicrobial activity against S. aureus, E. coli, P. aeruginosa, and C. albicans. The phytochemical screening revealed coumarins, terpenes/sterols, and flavonoids in the ethanolic extract. In addition, the coumarin identified as ayapin was isolated from this extract. We also performed in silico prediction of potential biological activities and targets for compounds previously found in A. fastigiatum. Several predictions were confirmed both retrospectively and prospectively by experimental results described here or elsewhere. Some activities described in the in silico target fishing approach were validated by the ethnopharmacological use and known biological properties. Some new activities and/or targets were predicted and could guide future studies. These results suggest that A. fastigiatum can be an interesting source of substances with antiparasitic and antimicrobial activities.

Lactococcus lactis FNBPA+ (pValac:e6ag85a) Induces Cellular and Humoral Immune Responses After Oral Immunization of Mice.

The development of a new vaccine strategy against tuberculosis is urgently needed and has been greatly encouraged by the scientific community worldwide. In this work, we constructed a lactococcal DNA vaccine based on the fusion of two Mycobacterium tuberculosis antigens, ESAT-6 and Ag85A, and examined its immunogenicity. The coding sequences of the ESAT-6 and Ag85A genes were fused and cloned into the eukaryotic expression pValac vector, and the functionality of the vector was confirmed in vitro. Then, L. lactis FnBPA+ (pValac:e6ag85a) was obtained and used for oral immunization of mice. This strain induced significant increases in IFN-γ, TNF-α, and IL-17 cytokines in stimulated splenocyte cultures, and significant production of antigen-specific sIgA was observed in the colonic tissues of immunized mice. We demonstrated that L. lactis FnBPA+ (pValac:e6ag85a) generated a cellular and humoral immune response after oral immunization of mice. The strategy developed in this work may represent an interesting DNA mucosal vaccine candidate against tuberculosis, using the fusion of two highly immunogenic antigens delivered by safe lactic acid bacteria.

Reverse and structural vaccinology approach to design a highly immunogenic multi-epitope subunit vaccine against Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a pathogen that resides in the upper respiratory tract of healthy individuals, maintaining a commensal relationship with its host. However, the virulent form may be the etiology of pneumonia, meningitis, bacteremia, and other respiratory tract infections. Streptococcal diseases are preventable by vaccination; but currently available vaccines have some drawbacks, especially due to the high capsule variability of streptococci strains. Thus, an effective prevention strategy continues to be the focus of extensive research. In our work, several bioinformatics tools were used to identify immunogenic peptides from a selected pool of 46 conserved proteins from Streptococcus pneumoniae. In silico analysis showed that 10 proteins had epitopes with affinity for B and T lymphocytes, which were present in at least 26 different pathogens serotypes and were considered promiscuous. The multi-epitope protein, designated HC44, was designed based on these epitopes and specific linkers to improve stability and exposure to T lymphocytes. The recombinant HC44 protein was expressed in E.coli and Swiss-Webster mice were immunised by intraperitoneal injection. Immunisation with the multi-epitope HC44 protein resulted in the production of very high levels of IgG with title superior to 1/1.200.000. However, subtype IgG was highly unbalanced toward IgG1 and no protection was afforded after challenge with S.pneumoniae in a sepsis model. Thus, our strategy has been effective in constructing a highly antigenic protein but novel immunisation strategies should be investigated to reorient the immune system toward a protective response.

Biochemical and functional properties of a new l-amino acid oxidase (LAAO) from Micrurus lemniscatus snake venom.

This study reports the purification of ML-LAAO, a new LAAO from the venom of Micrurus lemniscatus snake (ML-V), using size exclusion chromatography. ML-LAAO is a 69-kDa glycoprotein that represents ~2.0% of total venom proteins. This enzyme exhibited optimal activity at pH 8.5, displaying high specificity toward hydrophobic l-amino acids. MALDI TOF/TOF and Blast analysis identified internal segments in ML-LAAO that share high sequence identity with homologous snake venom LAAOs. Western blot analysis on two-dimensional SDS-PAGE of ML-V, using anti-LAAO revealed the presence of ML-LAAO isoforms (pI 6.3-8.9). ML-LAAO blocked aggregation induced by collagen on washed platelets in a rather weak manner, it did not, however, inhibit platelet aggregation induced by ADP on platelet-rich plasma. In addition, this enzyme displayed in vitro antibacterial activity against Staphylococcus aureus (MIC/MBC of 0.39 µg/mL) and in vitro leishmanicidal action against Leishmania amazonensis and L. chagasi (IC50 values of 0.14 and 0.039 µg/mL, respectively). These activities were significantly reduced by catalase, suggesting that hydrogen peroxide production is involved in some way. The data presented here revealed that ML-LAAO has bactericidal and leishmanicidal effects, suggesting that it may have therapeutic potential.

A New Broad Range Plasmid for DNA Delivery in Eukaryotic Cells Using Lactic Acid Bacteria: In Vitro and In Vivo Assays.

Lactococcus lactis is well documented as a promising candidate for development of novel oral live vaccines. It has been broadly engineered for heterologous expression, as well as for plasmid expression vector delivery, directly inside eukaryotic cells, for DNA vaccine, or as therapeutic vehicle. This work describes the characteristics of a new plasmid, pExu (extra chromosomal unit), for DNA delivery using L. lactis and evaluates its functionality both by in vitro and in vivo assays. This plasmid exhibits the following features: (1) a theta origin of replication and (2) an expression cassette containing a multiple cloning site and a eukaryotic promoter, the cytomegalovirus (pCMV). The functionality of pExu:egfp was evaluated by fluorescence microscopy. The L. lactis MG1363 (pExu:egfp) strains were administered by gavage to Balb/C mice and the eGFP expression was monitored by fluorescence microscopy. The pExu vector has demonstrated an excellent stability either in L. lactis or in Escherichia coli. The eGFP expression at different times in in vitro assay showed that 15.8% of CHO cells were able to express the protein after transfection. The enterocytes of mice showed the expression of eGFP protein. Thus, L. lactis carrying the pExu is a good candidate to deliver genes into eukaryotic cells.

Nonclassically secreted proteins as possible antigens for vaccine development: a reverse vaccinology approach.

Reverse vaccinology strategies have already been applied to a variety of microorganisms and have contributed significantly to vaccine development. However, most of the studies focused on an individual organism or on proteins with signature sequence motifs commonly found in known secreted proteins from bacteria. In this work, we applied a reverse vaccinology strategy based on conservation, virulence, and nonclassically surface exposure criterions to identify potential antigens in two microorganisms with significant degree of genomic plasticity among isolates (Streptococcus pneumoniae and Leptospira spp.), which imposes a major limitation to the production of a multistrain component vaccine. PSORTb 3.0.2 was run to predict the subcellular localization of the proteins. OrthoMCL was run to identify groups of the most conserved proteins between strains. Virulence prediction was done for the most conserved proteins, and SecretomeP was run to predict the nonclassically secreted proteins among the potential virulence factors. Based on the above criteria, we identified 37 proteins conserved between 16 genomes of S. pneumoniae and 12 proteins conserved between 5 leptospiral genomes as potential vaccine candidates.

Antigenic Peptides Capable of Inducing Specific Antibodies for Detection of the Major Alterations Found in Type 2B Von Willebrand Disease.

Von Willebrand disease (VWD) is an inherited hemorrhagic disorder promoted by either quantitative or qualitative defects of the von Willebrand factor (VWF). The disease represents the most common human coagulopathy afflicting 1.3% of the population. Qualitative defects are subdivided into four subtypes and classified according to the molecular dysfunction of the VWF. The differential diagnosis of the VWD is a difficult task, relying on a panel of tests aimed to assess the plasma levels and function of the VWF. Here, we propose biochemical approaches for the identification of structural variants of the VWF. A bioinformatic analysis was conducted to design seven peptides among which three were representatives of specific amino acid sequences belonging to normal VWF and four encompassed sequences found in the most common VWD subtype 2B. These peptides were used to immunize mice, after which, peptide-specific immunoglobulins were purified. This resulted in four Ig preparations capable of detecting alterations in the subtype 2B VWD plus additional three antibody fractions targeting the normal VWF. The panel of antibodies could serve many applications among them (1) assessment of VWF: antigen interaction, (2) VWF multimer analysis, and (3) production of monoclonal antibodies against VWF for therapeutic purposes as in thrombotic thrombocytopenic purpura.

Immune response and protective efficacy of S9 ribosomal protein of Streptococcus pneumoniae in a model of sepsis.

Vaccination is the most promising strategy to reduce the incidence of pneumococcal infection. Although there are vaccines available, all of them are based on polysaccharide antigens (conjugated or not). In addition to their high cost, those vaccines do not cover all serotypes. To overcome these hindrances, we evaluated the immunogenicity and the protective efficacy of the S9 ribosomal protein of Streptococcus pneumoniae with the aim of developing a protein-based vaccine in the future. The gene encoding the S9 ribosomal protein was cloned in pET21-a expression vector, and the recombinant S9 protein was used to immunize mice. Significantly higher levels of anti-S9 immunoglobulin G were achieved (with predominance of immunoglobulin G1) in comparison with the control. Antibodies elicited against S. pneumoniae protein extract in rabbit recognized the recombinant S9 protein by Western blot, thus demonstrating its immunogenicity. Moreover, mice immunized with recombinant S9 protein and challenged with a virulent strain of S. pneumoniae presented a significant reduction of bacteremia after 24 h of infection as compared with the control. However, in the S9-immunized mice the onset of death was insignificantly delayed, but all of them died by the fourth day postinfection.

Evaluation of water-in-oil-in-water multiple emulsion and microemulsion as potential adjuvants for immunization with rabies antigen.

Water-in-oil-in-water (W/O/W) multiple emulsions and microemulsions have been studied as potential candidates to be formulated as adjuvants. In this work their application as adjuvants for rabies virus immunization was studied. The humoral response, the effective dose 50 and histology for the developed formulations were evaluated in mice and compared with those from traditional adjuvants. The microemulsion and W/O/W multiple emulsion adjuvants developed were able to induce humoral response in mice and the serum showed good in vivo protection. Compared to the other adjuvants evaluated, microemulsion was shown to be the best candidate for rabies immunization as it presented good potency against the virus and did not appear to cause any local reaction.